Nova Patents
CA2280997A1

Dna-based transposon system for the introduction of nucleic acid into dna of a cell

Abstract

This invention relates to a system for introducing nucleic acid into the DNA of a cell. The system includes the use of a member of the SB family of transposases (SB) or nucleic acid encoding the transposase and a nucleic acid fragment that includes a nucleic acid sequence with flanking inverted repeats. The transposase recognizes at least a portion of an inverted repeats and incorporates the nucleic acid sequence into the DNA. Methods for use of this system are discussed.

CA2280997A1, drawing sheet 1
Sheet 1 of 20

Term

Term ended

Projected expiry passed 11 March 2018, 8.5 years ago.

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101 claims: 13 independent, 88 dependent

  1. 1
    What is Claimed is:1. A nucleic acid fragment comprising: a nucleic acid sequence positioned between at least two inverted repeats that bind to an SB protein, wherein the nucleic acid fragment is capable of integrating into DNA in a ceil.
  2. 21
    The nucleic acid fragment of claim I wherein the direct repeat has at least an 80% nucleic acid sequence identity to SEQ ID NO:10. 30
  3. 22
    A gene transfer system to introduce DNA into the DNA of a cell comprising:a nueteio acid fragment comprising a nueteie acid sequence positioned between at least two inverted rcpcatswhcrcin the inverted repeats can bind to an SBprotein and wherein the nucleic acid fragment is capable of integrating into DNA of a cell;and CA 02280997 1999-08-13 a nucleic acid fragment comprising a nucleic acid sequence positioned between at least two inverted repeats that bind to an SB protein, wherein the nucleic acid fragment is capable of integrating into DNA of a cell;and a transposase or nucleic acid encoding a transposase, wherein the transposase is an SB protein with an amino acid sequence sharing at least an 80% identity to SEQ ID NO:1.
  4. 48
    Nucleic acid encoding an SB protein, wherein the nucleic acid encodes a protein comprising SEQ ID NO:1 or a protein comprising an amino acid sequence with at least 80% identity to SEQ ID NO: I.
  5. 62
    An SB protein comprising the amino acid sequence of SEQ ID NO:1. 10
  6. 63
    A method for producing a transgenic animal comprising the steps of:introducing a nucleic acid fragment and a transposase into a pluripotent or totipotent cell wherein the nucleic acid fragment comprises a nucleic acid Sequence positioned between at least two inverted repeats that bind to an SB protein, said nucleic acid fragment being capable of integrating into DNA in a 15 cell, and wherein the transposase is an SB protein having an amino acid sequence identity of least 80% to SEQ ID NO: 1;and growing the cell into an animal. •t 20
  7. 68
    A method for introducing nucleic acid into DNA in a cell comprising the step of:introducing into a cell a nucleic acid fragment comprising a nucleic acid sequence positioned between at least two inverted repeats that bind to an SB y protein, wherein the nucleic acid fragment is capable-of integrating into DNA in a cell in the presence of an SB protein.
  8. 85
    A protein comprising the following characteristics:an ability to catalyze the integration of nucleic acid into DNA of a cell;capable of binding to the inverted repeat sequence of SEQ ID NOS :4 or 5;and 10 80% amino acid sequence identity to SEQ ID NO: 1. ι .(
  9. 86
    A protein comprising the following characteristics:transposase activity;a molecular weight range of about 35 kD to about 40 kD on about a 10% J 5 SDS-polyacrylamide gel;and I an nuclear localizing domain sequence, a DNA binding domain and a catalytic domain î wherein the protein has at least about five-fold improvement in the rate for ' introducing a nucleic acid fragment into the nucleic acid of a cell as compared to 20 the level obtained by non-homologous recombination. j
  10. 87
    A method for mobilizing a nucleic acid sequence in a cell comprising the steps of:introducing the protein of claims 84 or 85 into a cell housing DNA 25 containing the nucleic acid fragment according to claim 1, wherein the protein mobilizes the nucleic acid fragment from a first position within the DNA of a cell to a second position within the DNA of the cell.
  11. 92
    A method for identifying a gene in a genome of a cell comprising the steps of:ί ι introducing a nucleic acid fragment and an SB protein into a ceil, wherein the nucleic acid fragment comprises a nucleic acid sequence positioned between at least two inverted repeats that bind to the SB protein and wherein the nucleic acid fragment is capable of integrating into DNA in a cell in the presence of the -I SB protein;ι digesting the DNA of the cell with a restriction endonuclease capable of cleaving the nucleic acid sequence;ί identifying the inverted repeat sequences;sequencing the nucleic acid close to the inverted repeat sequences to obtain DNA sequence from an open reading^ frame;and comparing the DNA sequence with sequence information in a computer database.
  12. 95
    A stable transgenic vertebrate line comprising a gene operably linked to a 5 promoter, wherein the gene and promoter are flanked by inverted repeats that bind to an SB protein.
  13. 100
    A protein with transposase activity that can bind to one or more of the following sequences:SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ED NO:9, or SEQ ED NO: 10. 25 101. An antibody capable of specifically binding to an SB protein. J. CA 02280997 1999-08-13 WO 98/40510 PCT/US98/04687 ] A. IR/DR NLS D DNA-recognition ΟΙΛ « r«) IA t Ifc G-rich c v r» catalytic domain c* j I BE 340 IR/DR «·- «μ β «oo η co *r w b- eooceù n ** γμ rt κ » n kf» 01»»»·^·»·«- ** v - »- »-»- — cj *x AMS J u t tv SB10 11—restored ORF restored NLSactivrty restored DNAbinding activity restored integration activity 1/12 CA 02280997 1999-08-13 WO 98/40510 PCT/US98/04687 1 ATGGGAAAA TCAAAAGAAA TCAGCCAAGA CCTCAGAAAA TACCCTTTT AGTTTTCTTT AGTCGGTTCT GGAGTCTTTT 51 AAAATTGTAG ACCTCCACAA GTCTGGTTCA TCCTTGGGAG CAATTTCCAA TTTTAACATC TGGAGGTGTT CAGACCAAGT AGGAACCCTC GTTAAAGGTT
  14. 101
    101 ACGCCTGAAA GTACCACGTT CATCTGTACA AACAATAGTA CGCAAGTATA TGCGGACTTT CATGGTGCAA GTAGACATGT TTGTTATCAT GCGT7CATAT 151 AACACCATGG GACCACGCAG CCGTCATACC GCTCAGGAAG GAGACGCGTT TTGTGGTACC CTGGTGCGTC GGCAGTATGG CGAGTCCTTC CTCTGCGCAA 201 CTGTCTCCTA GAGATGAACG TACTTTGGTG CGAAAAGTGC AAATCAATCC GACAGAGGAT CTCTACTTGC ATGAAACCAC GCTTTTCACG TTTAGTTAGG 251 CAGAACAACA GCAAAGGACC TTGTGAAGAT GCTGGAGGAA ACAGGTACAA GTCTTGTTGT CGTTTCCTGG AACACTTCTA CGACCTCCTT TGTCCATGTT 301 AAGTATCTAT ATCCACAGTA AAACGAGTCC TATATCGACA TAACCTGAAA TTCATAGATA TAGGTGTCAT TTTGCTCAGG ATATAGCTGT ATTGGACTTT 351 GGCCGCTCAG CAAGGAAGAA GCCACTGCTC CAAAACCGAC ATAAGAAAGC CCGGCGAGTC GTTCCTTCTT CGGTGACGAG GTTTTGGCTG TATTCTTTCG 401 CAGACTACGG TTTGCAACTG CACATGGGGA CAAAGATCGT ACTTTTTGGA GTCTGATGCC AAACGTTGAC GTGTACCCCT GTTTCTAGCA TGAAAAACCT 451 GAAATGTCCT CTGGTCTGAT GAAACAAAAA TAGAACTGTT TGGCCATAAT CTTTACAGGA GACCAGACTA CTTTGTTTTT ATCTTGACAA ACCGGTATTA 501 GACCATCGTT ATGTTTGGAG GAAGAAGGGG GAGGCTTGCA AGCCGAAGAA CTGGTAGCAA TACAAACCTC CTTCTTCCCC CTCCGAACGT TCGGCTTCTT 551 CACCATCCCA ACCGTGAAGC ACGGGGGTGG CAGCATCATG TTGTGGGGGT GTGGTAGGGT TGGCACTTCG TGCCCCCAÇC GTCGTAGTAC AACACCCCCA 601 GCTTTGCTGC AGGAGGGACT GGTGCACTTC ACAAAATAGA TGGCATCATG CGAAACGACG TCCTCCCTGA CCACGTGAAG TGTTTTATCT ACCGTAGTAC 651 AGGAAGGAAA ATTATGTGGA TATATTGAAG CAACATCTCA AGACATCAGT TCCTTCCTTT TAATACACCT ATATAACTTC GTTGTAGAGT TCTGTAGTCA 701 CAGGAAGTTA'AAGCTTGGTC GCAAATGGGT CTTCCAAATG GACAATGACC GTCCTTCAAT TTCGAACCAG CGTTTACCCA GAAGGTTTAC CTGTTACTGG 751 CCAAGCATAC TTCCAAAGTT GTGGCAAAAT GGCTTAAGGA CAACAAAGTC GGTTCGTATG AAGGTTTCAA CACCGTTTTA CCGAATTCCT GTTGTTTCAG 801 AAGGTATTGG AGTGGCCATC ACAAAGCCCT GACCTCAATC CTATAGAAAA TTCCATAACC TCACCGGTAG TGTTTCGGGA CTGGAGTTAG GATATCTTTT 851 TTTGTGGGCA GAACTGAAAA AGCGTGTGCG AGCAAGGAGG CCTACAAACC AAACACCCGT CTTGACTTTT TCGCACACGC TCGTTCCTCC GGATGTTTGG 901 TGACTCAGTT ACACCAGCTC TGTCAGGAGG AATGGGCCAA AATTCACCCA ACTGAGTCAA TGTGGTCGAG ACAGTCCTCC TTACCCGGTT TTAAGTGGGT 951 ACTTATTGTG GGAAGCTTGT GGAAGGCTAC CCGAAACGTT TGACCCAAGT TGAATAACAC CCTTCGAACA CCTTCCGATG GGCTTTGCAA ACTGGGTTCA 1001 TAAACAATTT AAAGGCAATG CTACCAAATA CTAG. ATTTGTTAAA TTTCCGTTAC GATGGTTTAT GATC 2-A 2/12 CA 02280997 1999-08-13 WO 98/40510 PCT/ÜS98/04687 « a N ι ω C I—« w S ω J Λ s © © Ό ω λ• Μ CS H CO Ph Ο En ω Η pci !S Eh Pi Q Q Ot ω ι—ι ω co Q Eh Eh (7, Q O H Pi Pi Wl © Xi I ai ·*Μ U ! I i ω i •2 ’□ 1 3i O O !s S H CO Ni Q Q S a σ H a Eh ω Q pci CO Pi σ Pi CO Pi O Ni Pi H ! Ni Oh Ni u ω Ni Pi £ Pi Û s Eh CO H Ni H Q ω Es Es Pi CO a H Q S H Q H 0 0 W H Q CO Ot CO CO 't—I x—I Lf) o T-1 LO T—I rH O CN lO CN O O o Eh H H O O O O O0 3/12 CA 02280997 1999-08-13 WO 98/40510 PCT/US98/04687 CÛ 4/12 CA 02280997 1999-08-13 WO 98/40510 PCT/US98/04687 5/12 CA 02280997 1999-08-13 WO 98/40510 PCT/US98/04687 CA 02280997 1999-08-13 WO 98/40510 PCT/US98/O4687 O number of transformants per pétri dish 7/12 CA 02280997 1999-08-13 WO 98/40510 PCT/US98/04687 1 2 3 4 5 6 7 8 5 kb 3 kb 2 kb 8/12 CA 02280997 1999-08-13 WO 98/40510 PCT/ÜS98/04687 υ « o υ AJ Am O O fi. tjk Q 0 * υ σ. u υ a P « Λ g g ? b o υ « O\ υ D Q. ffl 9/12 CA 02280997 1999-08-13 WO 98/40510 PCT/US98/04687 v—A AAA A transposase mRNA microinjection into zebrafish embryos DNA isolation after 5 hrs incubation E. coli transformation;selection for Ap r /Km r colonies 10/12 CA 02280997 1999-08-13 WO 98/40510 PCT/US98/04687 CA 02280997 1999-08-13 WO 98/40510 PCT/US98/04687 i • Q. tn E c_ +□ o c QJ D re c o JZ ÛÛ a. 12/12
Independent claims14